Anti-PSGL-1 antibodies

ABSTRACT

Immunoglobulin chains or antibodies having light or heavy chain complementarity determining regions of antibodies that bind to P-Selectin Glycoprotein Ligand-1. Also disclosed are methods of inducing death of an activated T-cell and of modulating a T cell-mediated immune response in a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/399,516, filed Mar. 6, 2009, now abandoned, which is a continuationof U.S. patent application Ser. No. 11/125,837, filed May 10, 2005 nowU.S. Pat. No. 7,604,800, which claims priority to U.S. ProvisionalApplication No. 60/569,892, filed May 10, 2004, the contents of all ofwhich are incorporated by reference in their entirety.

BACKGROUND

Overly aggressive T cells often lead to unwanted immune responses,which, in turn, cause various disorders, e.g., autoimmune diseases,transplant rejection, allergic diseases, and T cell-derived cancers.Therefore, control of the aggressive T cells is critical in treatingsuch disorders. The activity of these cells can be contained byimmunosuppression or by induction of immunological tolerance. Analternative solution is induction of apoptosis, which is believed to beinvolved in removing unwanted cells, including overly aggressive Tcells. See, e.g., Kabelitz et al. (1993) Immunol Today 14, 338-340; andRaff (1992) Nature 356, 397-399.

SUMMARY

This invention relates to antibodies and their derivatives that induceapoptosis upon binding to P-Selectin Glycoprotein Ligand-1 (PSGL-1) onactivated T cells.

In one aspect, the invention features an immunoglobulin chain havingthree sequences that (i) contain, respectively, RSSQSIVHNDGNTYFE,KVSNRFS, and FQGSYVPLT (SEQ ID NOs: 1-3); (ii) contain, respectively,SFGMH, YINGGSSTIFYANAVKG, and YASYGGGAMDY (SEQ ID NOs: 4-6); (iii)contain, respectively, RASSTVNSTYLH, GSSNLAS, and QQYSGYPLT (SEQ ID NOs:7-9); (iv) contain, respectively, AYYIH, VNPNTGGTSYNPKFKG, andSGSPYYRYDD (SEQ ID NOs: 10-12); (v) contain, respectively,RSSQSIVNSNGNTYLE, KVSNRFS, and FQGSHVPWT (SEQ ID NOs: 13-15); or (vi)contain, respectively, TNAMNWVRQAPGKGLE, TYYADSVKD, and GGSYWYFDV (SEQID NOs: 16-18).

Each of the just-described six sets of sequences corresponds to thethree light or heavy chain complementarity determining regions (CDRs) ofan antibody that binds to PSGL-1, such as those of three mouse 15A7,43B6, and 9F9 antibodies described in the examples below. Shown beloware the light chains and heavy chain variable (V) regions of these threeantibodies (SEQ ID NOs: 19-26, the CDRs are underlined and highlighted):

Nucleic acid SEQ ID NO: 19 and amino acid SEQ ID NO: 27 (Mouse 15A7light chain V region):

Nucleic acid SEQ ID NO: 20 and amino acid SEQ ID NO: 28 (Mouse 15A7heavy chain V region):

Nucleic acid SEQ ID NO: 21 and amino acid SEQ ID NO: 29 (Mouse 43B6light chain V region):

Nucleic acid SEQ ID NO: 22 and amino acid SEQ ID NO: 30 (Mouse 43B6heavy chain V region):

Nucleic acid SEQ ID NO: 23 and amino acid SEQ ID NO: 31 (Mouse 9F9 lightchain V region):

Nucleic acid SEQ ID NO: 24 and amino acid SEQ ID NO: 32 (Mouse 9F9 heavychain V region):

As an antibody's antigen-binding specificity is determined by its lightand heavy chain CDRs, the above-described CDRs can be used to generateantibody derivatives that retain the antigen-binding specificity.Examples of antibody derivatives include chimeric antibodies, humanizedantibodies, and their functional equivalents. Shown below are the lightchain V region (SEQ ID NO: 25) and heavy chain V region (SEQ ID NO: 26)of a humanized 15A7 antibody, which include SEQ ID NOs: 1-3 and SEQ IDNOs: 4-6, respectively:

SEQ ID NO: 25 (humanized 15A7 light chain V region):

SEQ ID NO: 26 (humanized 15A7 heavy chain V region):

This invention also features an isolated nucleic acid having a sequencethat encodes one of the above-described immunoglobulin chains. The term“antibody” or “immunoglobulin chain” refers to an isolated polypeptide,i.e., a polypeptide that has been substantially separated from otherproteins, lipids, and nucleic acids with which it is naturallyassociated. The polypeptide can constitute at least 50, 70, or 95% bydry weight of the purified preparation. An “isolated nucleic acid”refers to a nucleic acid the structure of which is not identical to thatof any naturally occurring nucleic acid or to that of any fragment of anaturally occurring genomic nucleic acid. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. The nucleic acid of this invention can be used toexpress a polypeptide of this invention. For this purpose, one canoperatively link the nucleic acid to suitable regulatory sequences togenerate an expression vector.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked, and also capable ofautonomous replication or integration into a host DNA. Examples includea plasmid, cosmid, and viral vector. A vector of this invention includesa nucleic acid in a form suitable for expression of the nucleic acid ina host cell. Preferably, the vector includes one or more regulatorysequences operatively linked to the nucleic acid sequence to beexpressed. Examples of a regulatory sequence include promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences also include those that directconstitutive expression of a nucleotide sequence, as well astissue-specific regulatory and/or inducible sequences. The design ofsuch an expression vector is based on considerations including thechoice of the host cell to be transformed and the desired expressionlevel. An expression vector can be introduced into host cells to producea polypeptide of this invention. This invention also includes a hostcell that contains the above-described nucleic acid. A host cell refersto a cell containing an exogenous coding sequence or non-codingsequence. An exogenous sequence can be introduced into a cell by calciumphosphate transfection, DEAE-Dextran mediated transfection, orelectroporation. Suitable host cells include bacterial cells (e.g., E.coli, Bacillus subtilis, and Salmonella typhimurium), yeast cells (e.g.,Saccharomyces cerevisiae and Schizosaccharomyces pombe), plant cells(e.g., Nicotiana tabacum and Gossypium hirsutum), and mammalian cells(e.g., murine hybridoma cells, CHO cells, and 3T3 fibroblasts).

To produce an immunoglobulin chain of this invention, one can place ahost cell in a culture under conditions permitting expression of apolypeptide encoded by a nucleic acid described above, and isolate thepolypeptide from the culture. Alternatively, a nucleic acid of thisinvention can be transcribed and translated in vitro, for example, usingT7 promoter regulatory sequences and T7 polymerase.

Within the scope of this invention is an antibody. It is formed by afirst immunoglobulin chain and a second immunoglobulin chain, whichcontain, respectively, the light chain CDRs and heavy chain CDRs of themouse 15A7, 43B6, or 9F9 antibody mentioned above. Preferably, thisantibody is formed by the light and heavy chains of 15A7.

Also within the scope of this invention is another antibody that (i)binds specifically to P-Selectin Glycoprotein Ligand 1 withoutinterfering with binding between P-Selectin Glycoprotein Ligand 1 andP-Selectin and, (ii), upon binding to P-Selectin Glycoprotein Ligand 1on an activated T cell, induces the death of the T cell. In oneembodiment, this antibody binds specifically to human P-SelectinGlycoprotein Ligand 1.

Further within the scope of this invention is still another antibodythat binds specifically to amino acid residues 115-126 of mature humanP-Selectin Glycoprotein Ligand 1. Preferably, the antibody bindsspecifically to amino acid residues 117-123. More preferably, it bindsspecifically to amino acid residues 119-121, a consensus sequence amongall tested epitopes. Indeed, mutation of one or more of these threeamino acid residues abolishes antibody binding. In one example, thisantibody, upon binding to P-Selectin Glycoprotein Ligand 1 on anactivated T cell, induces the death of the activated T cell.

In one embodiment, one of the two antibodies mentioned immediately aboveis formed by a light chain and a heavy chain that contain, respectively,SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6 (e.g., SEQ ID NOs: 19 and 20, or SEQID NOs: 25 and 26).

In a further aspect, the invention features a method of inducing deathof an activated T cell. The method includes contacting one of the threeantibodies described above with an activated T cell, in which binding ofthe antibody to the activated T cell induces cell death.

The invention also features a method of modulating a T cell-mediatedimmune response in a subject. The method includes (1) identifying asubject having or as being at risk of having a condition related to anexcessive T cell-mediated immune response and (2) administering to thesubject an effective amount of one of the three antibodies describedabove. An “excessive T cell-mediated immune response” refers to aresponse caused by an excessive level of activated T cells. An excessivelevel refers to (1) a level higher than a normal level, and (2) a levelhigher than desired in an individual, even though it is not greater thana normal level. Examples of the condition include an inflammatorydisease, an autoimmune disease, an allergic disease, or a T cell cancer,as well as the situation in which a subject has received or iscontemplated to receive an allogeneic or xenogeneic transplant.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other features, objects, andadvantages of the invention will be apparent from the detaileddescription.

DETAILED DESCRIPTION

This invention is based, at least in part, on an unexpected discoverythat activated T cells can be induced to undergo apoptosis and bedepleted by binding of antibodies or their derivatives to PSGL-1 on theactivated cells. The antibodies and derivatives are useful for treatingconditions associated with an excessive or unwanted T cell-mediatedimmune response or T cell proliferation.

Accordingly, the invention features polypeptides that containimmunoglobulin light or heavy chain CDRs of anti-PSGL-1 antibodies, aswell as nucleic acids encoding them. Both the immunoglobulin chains andnucleic acids can be used to make the above-mentioned antibodies andderivatives.

An immunoglobulin chain of the invention can be obtained as a syntheticpolypeptide or a recombinant polypeptide. To prepare a recombinantpolypeptide, a nucleic acid encoding it can be linked to another nucleicacid encoding a fusion partner, e.g., Glutathione-S-Transferase (GST),6×-His epitope tag, M13 Gene 3 protein, or an immunoglobulin heavy chainconstant region. The resultant fusion nucleic acid can be introduced toa cell for protein expression. The fusion protein can be isolated fromthe host cell by methods well known in the art. The isolated fusionprotein can be further treated, e.g., by enzymatic digestion, to removethe fusion partner and obtain the recombinant polypeptide of interest.Alternatively, an immunoglobulin chain can be obtained from a suitablehost cell by activating endogenous expression of a nucleic acid encodingthe chain.

The amino acid composition of an immunoglobulin chain of the inventionmay vary without disrupting the ability of forming an antibody capableof binding to PSGL-1. For example, such a variant can contain one ormore conservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. Alternatively,mutations can be introduced randomly along all or part of a polypeptideof this invention, such as by saturation mutagenesis, and the resultantmutants can be screened for the ability of forming an antibody capableof binding to PSGL-1 to identify variants of this invention as describedbelow in the examples. Thus, as an example, the term “an immunoglobulinchain containing SEQ ID NO: 19” covers immunoglobulin chains containingvariants of SEQ ID NO: 19.

The above-described immunoglobulin chains and variants can be used tomake an antibody of this invention or its derivatives. An “antibody”includes intact molecules as well as fragments thereof, such as Fab,F(ab′)₂, Fv, scFv (single chain antibody), and dAb (domain antibody;Ward, et. al. (1989) Nature, 341, 544). A derivative of an antibodyrefers to a protein or a protein complex having a polypeptide variant ofthis invention. An antibody or derivative of this invention can be madeby co-expressing corresponding light and heavy chain CDRs-containingpolypeptides in a suitable host cell as described in the examples below.Alternatively, they can be made by methods known in the art of makingmonoclonal and polyclonal antibodies and fragments. See, e.g., Harlowand Lane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York.

To make an antibody of this invention, PSGL-1 or its antigenic fragmentcan be coupled to a carrier protein, such as KLH, mixed with anadjuvant, and injected into a host animal. Antibodies produced in thatanimal can then be purified by peptide affinity chromatography. Commonlyemployed host animals include rabbits, mice, guinea pigs, and rats.Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Useful human adjuvants include BCG (bacilleCalmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies, heterogeneous populations of antibody molecules,are present in the sera of the immunized subjects. Monoclonalantibodies, homogeneous populations of antibodies to a particularantigen, can be prepared using standard hybridoma technology. See, e.g.,Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J.Immunol. 6, 511; Kohler et al. (1976) Eur. J. Immunol. 6, 292; andHammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y. In particular, monoclonal antibodies can be obtained byany technique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in U.S. Pat. No.4,376,110; the human B-cell hybridoma technique (Kosbor et al. (1983)Immunol Today 4, 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80,2026) and the EBV-hybridoma technique (Cole et al. (1983) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Suchantibodies can be of any immunoglobulin class including IgG, IgM, IgE,IgA, IgD, and any subclass thereof. The hybridoma producing themonoclonal antibodies of the invention may be cultivated in vitro or invivo. The ability to produce high titers of monoclonal antibodies invivo makes it a particularly useful method of production.

In addition, techniques developed for the production of “chimericantibodies” can be used. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314, 452. A chimeric antibody is a moleculein which different portions are derived from different animal species,such as those having a variable region derived from a murine monoclonalantibody and a human immunoglobulin constant region. Alternatively,techniques described for the production of single chain antibodies (U.S.Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phagelibrary of single chain Fv antibodies. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge. Moreover, antibody fragments can be generatedby known techniques. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments that can be produced by pepsin digestionof an antibody molecule, and Fab fragments that can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Antibodies can alsobe humanized by methods described in the examples below or known in theart. For example, monoclonal antibodies with a desired bindingspecificity can be commercially humanized (Scotgene, Scotland; andOxford Molecular, Palo Alto, Calif.). Fully humanized antibodies, suchas those expressed in transgenic animals are within the scope of theinvention (see, e.g., Green et al. (1994) Nature Genetics 7, 13; andU.S. Pat. Nos. 5,545,806 and 5,569,825).

Also within the scope of this invention is a method of inducing death ofactivated T cells, e.g., by contacting activated T cells with anantibody of the invention in vitro, and by administering to a subject inneed thereof an effective amount of the antibody. Subjects to be treatedcan be identified as having or being at risk for having a conditionrelated to an excessive or unwanted T cell-mediated immune response,e.g., patients suffering from autoimmune diseases, transplant rejection,allergic diseases, or T cell-derived cancers. This method can beperformed alone or in conjunction with other drugs or therapy.

The term “treating” refers to administration of a composition to asubject with the purpose to cure, alleviate, relieve, remedy, prevent,or ameliorate a disorder, the symptom of the disorder, the disease statesecondary to the disorder, or the predisposition toward the disorder. An“effective amount” is an amount of the composition that is capable ofproducing a medically desirable result in a treated subject.

Exemplary diseases to be treated include diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, and psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosus,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type Idiabetes, inflammatory bowel diseases, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, interstitial lung fibrosis, graft-versus-hostdisease, cases of transplantation (including transplantation usingallogeneic or xenogeneic tissues) such as bone marrow transplantation,liver transplantation, or the transplantation of any organ or tissue,allergies such as atopic allergy, AIDS, and T cell neoplasms such asleukemias or lymphomas.

In one in vivo approach, a therapeutic composition (e.g., a compositioncontaining an antibody of the invention) is administered to the subject.Generally, the antibody is suspended in a pharmaceutically-acceptablecarrier (e.g., physiological saline) and administered orally or byintravenous infusion, or injected or implanted subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thesubject's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compositions available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the composition in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Also within the scope of this invention is a pharmaceutical compositionthat contains a pharmaceutically-acceptable carrier and an effectiveamount of an antibody of the invention. The pharmaceutical compositioncan be used to treat diseases described above. Thepharmaceutically-acceptable carrier includes a solvent, a dispersionmedium, a coating, an antibacterial and antifungal agent, and anisotonic and absorption delaying agent.

The pharmaceutical composition of the invention can be formulated intodosage forms for different administration routes utilizing conventionalmethods. For example, it can be formulated in a capsule, a gel seal, ora tablet for oral administration. Capsules can contain any standardpharmaceutically-acceptable materials such as gelatin or cellulose.Tablets can be formulated in accordance with conventional procedures bycompressing mixtures of the composition with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. The composition can also be administered in a form of a hardshell tablet or a capsule containing a binder, e.g., lactose ormannitol, a conventional filler, and a tableting agent. Thepharmaceutical composition can be administered via the parenteral route.Examples of parenteral dosage forms include aqueous solutions, isotonicsaline or 5% glucose of the active agent, or other well-knownpharmaceutically-acceptable excipient. Cyclodextrins, or othersolubilizing agents well known to those familiar with the art, can beutilized as pharmaceutical excipients for delivery of the therapeuticagent.

The efficacy of a composition of this invention can be evaluated both invitro and in vivo. See, e.g., the examples below. Briefly, thecomposition can be tested for its ability to induce death of activated Tcells in vitro. For in vivo studies, the composition can be injectedinto an animal (e.g., a mouse model) and its therapeutic effects arethen accessed. Based on the results, an appropriate dosage range andadministration route can be determined.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

EXAMPLE 1 Mouse Monoclonal Antibodies 15A7, 43B6, and 9F9

Generation of Anti-PSGL-1 Antibodies

Standard techniques were used to generate mouse monoclonal antibodiesthat specifically bound to human PSGL-1 (hCD162). More specifically,mice were immunized with membrane fraction of PHA-activated human Tcells and sacrificed to generate hybridoma cell lines. Supernatants fromresultant hybridoma cell lines were screened for binding to CHO cellsthat stably expressed hCD162. Those lines producing antibodies thatbound to hCD162-expressing CHO cells, but not the parental CHO cells,were identified, subcloned, and further analyzed as described below.

Among the lines identified were m152-15A7, m166-43B6, and m128-9F9. Theyproduced IgG1 antibodies 15A7, 43B6, and 9F9, respectively.Immunoblotting assay showed that these three antibodies pulled down fromlysate of activated T-cells a protein that could be detected byanti-hCD162 antibody (kp1-1, PharMingen, San Diego, Calif.).

The just-described three antibodies were tested for their abilities toinduce apoptosis of activated T cells. Culture supernatants containingmonoclonal antibodies secreted by the three hybridoma cell lines wererespectively incubated with either non-activated human T cells (Day 0)or in vitro activated human T cells (Day 7) for 6 hours. The cells werethen stained with annexin V and subjected to FACS analysis. CD3-positivecells were gated to ensure counting of either in vitro activated human Tcells or resting human T cells. The apoptotic cells were annexin Vstaining-positive. Table 1 summarizes the percentage of apoptotic Tcells among all of the T cells scanned.

TABLE 1 Percentage of apoptotic T cells Anti- m128- Un- Anti- m152-M166- Untreated myc 9F9 treated myc 15A7 43136 Day 0 4.17 6.67 5.8218.18 15.52 5.23 6.57 Day 7 12.63 13.36 28.71 24.18 23.08 51.66 49.44

These results indicate that mouse 15A7, 43B6, and 9F9 antibodies (1) arehCD162-specific and (2) can bind to human activated T cells and induceapoptosis of activated T cells, but not resting human T cells.

Apoptosis assay was also conducted on PHA-activated human peripheralblood mononuclear cells (PBMC). It was found that the antibodies onlyinduced apoptosis in activated T cells, but not in resting T cells, Bcells, or in neutrophils.

It is known that T cell-depleting antibodies, such as anti-CD3, are ableto induce production of soluble factors. Therapy using such antibodiesusually results in a deleterious cytokine syndrome. To test ifanti-PSGL-1 antibody also caused cytokine-associated side effects,freshly isolated human PBMC were cultured with 15A7 for 24, 48, or 72hours. The levels of cytokines in the supernatant were then determined.Considerable amounts of IL-2, TNF-α, and IFN-γ were produced inPHA-activated PBMC (positive control), while levels of these cytokinesfrom 15A7-treated cells were not detectable. These results supportedthat anti-PSGL-1 has no or little effect on resting peripheral bloodcells, in both aspects of apoptotic induction and cell activation.

Since the above-described antibodies selectively induce apoptosis ofactivated T cells without causing adverse effects on resting T or otherimmune cells, administration of them to a subject is unlikely to resultin lymphopenia or broad immunodeficiency like anti-CD3 orimmunosuppressant does.

Epitope Mapping of Anti-CD 162 Antibodies

To map the binding epitopes of mouse 15A7, 43B6, and 9F9 on human CD162,a series of fusion proteins covering various regions of human CD162 wereexpressed and purified. Interactions between the fusion proteins andthese monoclonal antibodies were examined by sandwich enzyme-linkedimmunosorbent assay (ELISA).

Briefly, fragments covering various regions of human CD 162 gene wereexpressed as fusion proteins with human immunoglobulin gamma 1 heavychain constant region in E. coli. cDNA encoding the human immunoglobulingamma 1 heavy chain constant region was amplified by PCR with primershaving a BglII site and a BamHI site. The PCR product was cut by BglIIand BamHI, and subcloned into a pET-32a vector (Novagen®) that had beendigested by the same enzymes. Then, cDNAs encoding various regions ofhCD 162 were amplified by PCR with primers having an NdeI site at the 5′end and a BglII site at the 3′ end. The PCR products were cut by thecorresponding enzymes and in frame fused to the sequence encoding thehuman immunoglobulin gamma 1 heavy chain constant region in the pET-32avector. Primers used in each construction are listed in Table 2, and thesequences of the primers are listed in Table 3.

TABLE 2 Names of primers used in each experiment For amplifyingsequences encoding: Forward primer Reverse primer E. coli expressedhCD162 fragments  42-119 AB1001 AB1005  42-80 AB1001 AB1008  61-99AB1003 AB1009  81-119 Ab1004 AB1005  42-70 AB1001 AB1007  42-60 AB1001AB1006  50-80 AB1002 AB1008  50-70 AB1002 Ab1007  42-319 AB1001 Ab1010115-126 AB1022 AB1023 115-126EtoR AB1024 AB1025 V region of cDNAs lightchain AB1058 AB1059 heavy chain AB1058 AB1060 Mammalian expressed hCD162fragments  1-119 AB1011 AB1013  1-319 AB1011 AB1012 110-319 AB1058AB1059  94-148 AB1020 AB1021 119-222 AB1018 AB1019 174-269 AB1016 AB1017214-317 AB1014 AB1015 Chimeric chains 15A7 light chain AB1030 AB103115A7 heavy chain AB1032 AB1033 9F9 light chain AB1026 A131027 9F9 heavychain AB1028 AB1029 43B6 light chain AB1034 AB1035 43B6 heavy chainAB1036 AB1037 Humanized chains 15A7 light chain AB1048 AB1057 15A7 lightchain 1st pair AD1049 AB1050 15A7 light chain 2nd pair AB1051 AB105215A7 light chain 3rd pair AB1053 AB1054 15A7 light chain 4th pair AB1055Ab1056 15A7 heavy chain AB1038 AB1047 15A7 heavy chain 1st pair AB1039AB1040 15A7 heavy chain 2nd pair AB1041 AB1042 15A7 heavy chain 3rd pairAB1043 AB1044 15A7 heavy chain 4th pair AB1045 AB1046

TABLE 3 Primer sequences SEQ ID Name Sequence NO: AB1001cccgggacCATATGcaggccaccgaatatgagtacc 39 AB1002tatgagCATATGgattatgatttcctgccagaaacgg 40 AB1003aaacggagCATATGgaaatgctgaggaacagcactgacacc 41 AB1004aacccctCATATGaccactgtggagcctgctgcaaggcg 42 AB1005gtggtcAGATCTtccatagctgctgaatccgtggacagg 43 AB1006GTTCCTCAGATCTTCTGGAGGCTCCGTTTCTGGCAGG 44 AB1007AGGCCCAAGATCTGGAGTGGTGTCAGTGCTGTTCCTC 45 AB1008ggctccAGATCTgtagactcaggggttccaggccc 46 AB1009gtggtcAGATCTgtgactgcccctcctgcatccaggcc 47 AB1010GCCAGCAGATCTTGCTTCACAGAGATGTGGTCTGGGG 48 AB1011cgcggatccatgcctctgcaactcctcctgttgc 49 AB1012GCCAGCCTCGAGCTTCACAGAGATGTGGTCTGGGG 50 AB1013GGTCTGctcgagCATAGCTGCTGAATCCGTGGACAGGTTC 51 AB1061agacaggccaccgaagggaacctgtccacg 52 AB1062 cgtggacaggttcccttcggtggcctgtct53 AB1014 ccgctcgagcgccaagattaggatggc 54 AB1015cgggatccactcaaaccacagccatgg 55 AB1016 ccgctcgagtggtagtaggttccatgg 56AB1017 cgggatcaactcaacccacaggcctg 57 AB1018 ctgtgcctcgagggctgtggtttgagtg58 AB1019 cgggatccatggagatacagaccactcaac 59 AB1020cgggatccgatgcaggaggggcagtcac 60 AB1021 ggccgtcactcgagttgtctgtgcctc 61AB1022 TatgGATTCAGCAGCTATGGAGATACAGACCACTCAACCAgcA 62 AB1023GATCTgcTGGTTGAGTGGTCTGTATCTCCATAGCTGCTGAATCCA 63 AB1024TatgGATTCAGCAGCTATGCGGATACAGACCACTCAACCAgcA 64 AB1025GATCTgcTGGTTGAGTGGTCTGTATCCGCATAGCTGCTGAATCCA 65 AB1026CTAGTCTAGATGACCCAAACTCCACTCTCCC 66 AB1027CTAGTCTAGAATTAGGAAAGTGCACTTAGCATCAGCCCGTTTGATTTCC 67 AB1028TAACATtctagATGCTGTTGGGGCTGAAGTGGG 68 AB1029GGATAGTCTAGAGGTTGTGAGGACTCACCTGAGGAGACGGTGACCGTGG 69 AB1030CTAGTCTAGATGGAGACAGACACACTCCTGTTATGGG 70 AB1031CTAGTCTAGAATTAGGAAAGTGCACTTTTTCCAGCTTGGTCCCCCCTCC 71 AB1032CTAGTCTAGATGGACTCCAGGCTCAATTTAGTTTTCC 72 AB1033CTAGTCTAGAGGTTGTGAGGACTCACCTGAGGAGACGGTGACTGAGGttcc 73 AB1034CTAGTCTAGATGGATTTTCTGGTGCAGATTTTCAGC 74 AB1035CTAGTCTAGAATTAGGAAAGTGCACTTAGCATCAGCCCGTTTCAGCTCC 75 AB1036CTAGTCTAGATGGAATGGAGCTGGGTCTTTCTC 76 AB1037CTAGTCTAGAGGTTGTGAGGACTCACCAGCTTCCAGTGGATAGACTGATGG 77 AB1038TCTATCTAGATGAACTTCGGGTCCAGCTTGATTTTCCTTGTCCTTGTTTTA 78 AAAGGTGTCCAGTGAB1039 CCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGCAACTGGTGGAGTCTGGGGGA 79GGCTTAGTGCAGCCTGG AB1040CTGAAAGTGAATCCAGAGGCTGCACAGGAGAGTCTCAAGCTTCCTCCAGGC 80 TGCACTAAGCCTCCAB1041 GCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTC 81CAGGGAAGGGACTCGAG AB1042GCATAGAAGATGGTACTACTGCCACCATTAATGTATGCGACCCACTCGAGT 82 CCCTTCCCTGGAGCCAB1043 GTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCA 83GAGATAATGCC AB1044 CCTCAGCCCTCAGAGAATTCATTTGCAGGTACAGGGTGTTCTTGGCATTAT84 CTCTGGAGATGG AB1045GAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGC 85 TAGTTACGGAGGAB1046 CTGTGACCAGGGTGCCTTGGCCCCAATAGTCCATAGCACCCCCTCCGTAAC 86 TAGCATATCAB1047 ACCCTCTAGAGGTTGTGAGGACTCACCTGAGGAGACTGTGACCAGGGTGCC 87 TTGGCCAB1048 TCTATCTAGATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGG 88 GTTCCAGGCAB1049 GCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTCAGATGACCCAATCTCC 89 GAGCTCTTTGAB1050 GATCTGCAGGTGATAGTGACCCTATCCCCTACAGACGCAGACAAAGAGCTC 90 GGAGATTGGAB1051 CACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACAC 91CTATTTTGAATG AB1052 GATGAGAAGCTTGGGTGCCTTTCCTGGTTTCTGTTGGTACCATTCAAAATA92 GGTGTTTC AB1053 GCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCA93 TCCAGGTTTAGTGGC AB1054GCAGAGAAGAGATGGTGAGGGTGAAGTGTGTCCCAGACCCACTGCCACTAA 94 ACCTGGATGG AB1055CTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTT 95 CAAG AB1056CCTTGGTGCCTTGACCGAACGTGAGAGGAACATATGAACCTTGAAAACAGT 96 AATAGG AB1057ACCCTCTAGAATTAGGAAAGTGCACTTACGTTTGATTTCCACCTTGGTGCC 97 TTGACCG AB1058TATATCTAGAATTCCCCCCCCCCCCCCCCC 98 AB1059TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC 99 AB1060TATAGAGCTCAAGCTTCCAGTGGATAGAC(C/A/T)GATGGGG(C/G) 100 TGT(C/T)GTTTTGGC

The above-described expression constructs were transformed intoEscherichia coli strain BL21 (DE3). The transformed cells were harvestedafter 6 hours of IPTG (2 mM) induction and resuspended in PBS. After thecells were sonicated and spun down at 14,000 g for 10 minutes, theresultant supernatants were collected for purification of the fusionproteins. More specifically, the supernatants were first incubated withprotein G or protein A beads for 3 hours at 4° C. The beads were thenspun down at 3,000 g and washed with washing buffer I (0.05% Triton®X-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl, 1 mM CaCl₂ and 1 mg/ml OVA)and washing buffer II (0.05% Triton® X-100, 50 mM Tris-HCl, pH 8.5 and1.50 mM of NaCl) for 5 times each. Bound proteins were then eluted withan elution buffer containing 0.1 M of glycine-HCl, pH 2.7 andneutralized with 1 M Tris-HCl, pH 8.6. All purified fusion proteins werequantified by Bio-Rad® protein assay (Bio-Rad® Laboratories, Cat. No.500-0006) and verified by SDS-PAGE.

A sandwich ELISA was conducted to study the interaction between thehCD162 fragments and each of 15A7, 9F9, and 43B6. 96-well microtiterplates were coated with goat anti-human IgG (Southern Biotechnology,Cat. No. 2040-01) antibody (2 μg/ml, 50 μl/well) overnight at 4° C.Plates were blocked by incubation with 0.25% of BSA in PBS (150 μl/well)for 1 hour at 37° C. The blocked plates were then incubated with fusionproteins containing various fragments of human CD162 (2 μg/ml) for 2hours at room temperature. After washing 4 times with PBS containing0.05% of Tween®20 (PBST), the plates were incubated with testingantibodies (2 μg/ml) for 1.5 hours at room temperature. Afterincubation, the plates were washed 4 times with PBST. 50 μl of 1 to 3000diluted goat anti-mouse IgG conjugated with alkaline phosphatase(Southern Biotechnology, Cat. No. 1031-04) was then added to each welland the plates were incubated for 1 hour at 37° C. Enzyme reaction wascarried out by adding 50 μl of an alkaline phosphatase substratesolution (1 alkaline phosphatase substrate tablet dissolved in 5 ml ofsubstrate buffer containing 0.012 M of Na₂CO₃, 0.16 M of NaHCO₃ and 1 mMof MgCl₂ at pH 8.6), and absorbance at 405 nm was determined.

It was found that 43B6 and 9F9 were able to interact with all fusionproteins containing residues 50 to 60 of mature human CD162, indicatingthat epitopes of 43B6 and 9F9 were located between residues 50-60.Unlike 9F9 and 43B6, 15A7 only bound to the fusion protein coveringresidues 42 to 319, but not the fusion protein covering residues 42-119,indicating that the epitope of 15A7 was located between residues 119 to319. The location of the epitope of 15A7 was then narrowed down tobetween residues 115 to 126. Change of one amino acid at position 120(Glu→Arg) diminished interaction between 15A7 and the fusion protein,indicating that the primary contacting domain of 15A7 on human CD162 islocated at or adjacent to position 120, and the residue Glu is essentialfor the interaction.

Fusion proteins covering various human CD162 regions were also expressedin mammalian cells and were tested for their interaction with 15A7.Fragments covering these regions were expressed as fusion proteins withhuman immunoglobulin gamma 1 heavy chain constant region in mammaliancells. First, the cDNA encoding human immunoglobulin gamma 1 heavy chainconstant region was inserted into a pcDNA3 vector (Invitrogen). Second,cDNAs encoding various regions of hCD162 were amplified by PCR withprimers introducing a BamHI site at the 5′ end and an XhoI site at the3′ end. These PCR products were cut by the corresponding enzymes andsubcloned into the human immunoglobulin gamma 1 heavy chain constantregion-containing pcDNA3 vector. The name and sequence for each primerare listed in Tables 2 and 3 above.

The just-described mammalian expression vectors were transientlytransfected into COS-7 cells by Lipofectamine™ 2000 (Invitrogen™, Cat.No. 11668-027) following manufacturer's guide. The transfected cellswere grown in ultra low-Ig medium (Invitrogen™, Cat. No. 16250-078). Theexpressed proteins were purified and subjected to sandwich ELISA in thesame manner described above.

The ELISA results show that only the fusion proteins containing residues94 to 148 were able to interact with 15A7. These results are consistentwith the idea that the epitope of 15A7 is located between residues 115to 126.

All of the above results indicate that the epitopes of 9F9, 43B6, and15A7 are protein-dependent, instead of carbohydratemodification-dependent, since all three antibodies bind bacteriallyexpressed fusion proteins. They also indicate that, although 15A7, 9F9,and 43B6 show similar properties in term of binding specificity andfunction of inducing apoptosis in activated T cells, they functionthrough different domains of human CD162 and behave differently.

EXAMPLE 2 Chimeric Antibodies 15A7, 43B6, and 9F9

Cloning of Light and Heavy Chain Variable Regions of Anti-CD162Antibodies

cDNAs encoding the light and heavy chain variable regions (V_(L) andV_(H)) of antibodies 15A7, 43B6, and 9F9 were amplified by an anchoredPCR method. The 3′ primers hybridized to the C regions and the 5′primers hybridized to G tails attached to the cDNA using terminaldeoxytransferase. The PCR fragments were cloned into a pCRII vector(Invitrogen™). Several independent clones for each chain were sequencedand compared. A sequence represented by the majority of the independentclones was picked. The translated amino acid sequence was then analyzedto confirm that the chosen sequence possessed the characteristics oftypical mouse light or heavy chain V region, and belonged to a specificsubtype. The complementarity determining regions (CDRs) were thenidentified by comparing the translated amino acid sequences withconsensus sequence of each subtype. The name and sequence for eachprimer used are listed in Tables 2 and 3 above. The deduced amino acidsequences of the light and heavy chain V regions of 15A7, 43B6, and 9F9(SEQ ID NOs: 19-24) are shown in Summary.

Chimeric Antibodies

To generate vectors for expressing chimeric antibodies, cDNAs encodingthe V_(L) and V_(H) regions of 15A7, 43B6, and 9F9 were amplified by PCRusing primers to include the 5′ signal peptide sequence and the 3′splice donor signal. The primers also introduced XbaI sites at both endsof the PCR products, which were then cut by XbaI enzyme and ligated intoXbaI-digested pVκ, pVg1, pVg2, or pVg4 vector. More specifically, theV_(L) region cDNAs of 15A7, 43B6, and 9F9 were subcloned into theplasmid pVκ. This plasmid contained a CMV promoter, and a sequenceencoding the human light chain constant region. The V_(H) region cDNAsof 15A7, 43B6 and 9F9 were subcloned into plasmids pVg1, pVg2, or pVg4.Each of the three plasmids had a CMV promoter. They also contained,respectively, the human heavy chain constant regions of IgG1, IgG2, andIgG4.

Each of the above-described light chain-encoding plasmids wasco-transfected with a heavy chain-encoding plasmid into COS-7 cells. Thesupernatants of the transfected cells were collected. Chimericantibodies in the supernatants were analyzed for the ability to bind tohuman CD162 and to induce apoptosis of activated T cells.

It was found that all chimeric antibodies made from 15A7, 43B6, and 9F9bound to Sp2/0 transfectants stably expressing human CD162, but not toparental Sp2/0 cells, indicating that they retained the humanCD162-binding ability specificity. Furthermore, it was found that thechimeric antibodies induced apoptosis in T cells that had been activatedfor 7 days, indicating that they retained this function of their mousecounterparts as well.

Humanized Antibodies

Mouse 15A7 was used to make humanized antibodies by grafting its CDRsonto a human framework. To retain binding affinity and specificity, itis essential to conserve the V region conformation when grafting theCDRs onto the human framework. To select a proper framework donor, theamino acid sequences of mouse 15A7 light and heavy chain V regions werecompared with those of 50 mouse antibodies that had been humanized.

It was found that a mouse antibody, mDREG-55, had high sequence homologyto mouse 15A7 V region in both light and heavy chains. Listed below is asequence alignment of mouse 15A7 against this mDREG-55 antibody (CDRsare highlighted):

Mouse DREG-55 is a monoclonal IgG1 antibody against L-selectin. Thesequences of mouse 15A7 V_(L) and V_(H) regions were respectively 64.3%(framework only: 73.8%) and 70% (framework only: 81.6%) homologous tothose of mouse DREG55. Humanized DREG-55 (HuDREG-55) had beenconstructed using framework sequences of V_(L) and V_(H) regions from ahuman antibody Gal. Therefore, to humanize mouse 15A7, the frameworksequences of human Gal light and heavy chains were used to replace thecounter parts of mouse 15A7.

The humanized 15A7 light and heavy variable regions were each assembledby 4 pairs of synthetic oligonucleotides (˜80 bases in length). Theoligonucleotides of each pair were overlapped by around 20 nucleotides.Nucleotide sequences were selected and synthesized to encode the proteinsequences of the humanized variable regions including signal peptides.The assembling and amplification of the genes were conducted in foursteps: (1) the four pairs of complementary oligonucleotides wereannealed and extended with Klenow fragment in 4 separate reactions; (2)the resulting 4 dsDNA fragments were mixed pair wise, denatured,reannealed, and extended in two separate reactions; (3) the resultingtwo dsDNA fragments were mixed, denatured, reannealed, and extended tocreate the final full-length dsDNA; and (4) the resulting DNA wasamplified by PCR with primers to introduce an XbaI site at both ends.The PCR fragment was then cut by XbaI and inserted into the respectiveXbaI-digested pVκ and pVg4 vectors. Then, at positions where theinteractions between CDR and the framework were considered important,the Ga1's residues were changed back into those of the mouse 15A7 (i.e.,I62V and D74H). Listed below are alignments of mouse 15A7 and humanized15A7 (Hu15A7) against mDREG-55, in which V62 and H74 are underlined.

Plasmids thus obtained encoded humanized 15A7 heavy and light chains.These plasmids were then co-transfected into COS-7 cells. The exhaustedsupernatants from cultured cells were then collected. Humanized 15A7 inthe supernatants was tested for its ability to bind to CHO transfectantsstably expressing hCD162 and to induce apoptosis in the T cellsactivated for 7 days. The results show that it retains these abilities.

Preparation of Chimeric and Humanized Antibodies

Cells producing humanized and chimeric antibodies were generated. Morespecifically, Sp2/0 cells (Sp2/0-Ag14; ATCC CRL 1581) were stablytransfected with the appropriate plasmids by electroporation using aGene Pulser® apparatus (Bio-Rad® Laboratories) at 360 V and 25 μFcapacitance according to the manufacturer's instructions. Beforetransfection, the plasmids were linearized by digesting with BamHIenzyme. All transfections were performed using 10⁷ cells in PBS and 20μg each of plasmid DNA. The cells from each transfection were platedinto two 96-well tissue culture plates. After 48 hours, a selectivemedium (DMEM 10% FBS/hypoxanthine/thymidine media supplement) and 1μg/ml mycophenolic acid was applied. Antibody-producing cells werescreened and isolated by examining the presence of antibody in theculture supernatant by ELISA.

Isolated cells were cultured in serum-free or low-Ig medium, and thecultured supernatant was collected. Antibodies were purified by passageover a column of staphylococcal protein A-Sepharose CL-4B. After washing5 times each with washing buffer I (0.05% Triton®X-100, 50 mM Tris-HCl,pH 8.5, 400 mM NaCl, 1 mM CaCl₂ and 1 mg/ml OVA) and washing buffer II(0.05% Triton®X-100, 50 mM Tris-HCl, pH 8.5 and 150 mM of NaCl), thebound antibodies were eluted with an elution buffer containing 0.1 M ofglycine-HCl, pH 2.7, and neutralized with 1 M Tris-HCl, pH 8.6.

Affinity Measurements

Binding affinities of the above-described mouse, chimeric, and humanized15A7 antibodies were determined by competitive binding.

Mouse 15A7 was biotinylated by an EZ-Link™ Sulfo-NHS-Biotin system(Pierce Biotechnology, Cat. No. 21217). Briefly, 0.5 mg (3.3×10⁻⁶nmoles) of mouse 15A7 was dissolved in 187 μl of PBS and mixed with6.8×10⁻⁵ nmoles of Sulfo-NHS-Biotin. The mixture was then incubated onice for 2 hours before free biotins were removed by dialyzing at 4° C.overnight against PBS. The Biotin-labeled mouse 15A7 thus obtained wasstored at 4° C. until use.

Sp2/0 transfectants stably expressing human CD162 were used as source ofhuman CD162 antigen. Biotin-labeled mouse 15A7 was used as tracer.Increasing amounts of competitor antibodies (mouse, chimeric, orhumanized 15A7) were mixed with 35 ng of Biotin-labeled mouse 15A7 andincubated with 1×10⁵ CD162-expressing Sp2/0 cells for 1.5 hours at 4° C.with constant shaking. After washing, secondary antibody,Streptavidin-PE (Becton Dickinson Immunocytometry System Inc. Cat. No.349023) was added to the mixture. After incubating for 45 minutes at 4°C., the cells were washed again, resuspended in 300 μl of PBS-1% of FBS,and subjected to FACS analysis.

It was found that the half-maximum competing concentration of mouse 15A7was 3.72 μg/ml while those of chimeric and humanized 15A7 were around5.71 μg/ml and 4.51 μg/ml, respectively. These results indicate that theaffinities of mouse, chimeric, and humanized 15A7 are comparable. Inother words, the binding affinity (Ka) for mouse 15A7 is 4.03×10⁷ M⁻¹while those for chimeric and humanized 15A7 are 2.62×10⁷ M⁻¹ and3.33×10⁷ M⁻¹, respectively.

Competition Analysis

Competition analysis was conducted to study interaction among theabove-described three mouse antibodies, PSGL-1, and P-selectin.

P-selectin is a major high-affinity ligand for PSGL-1 on mostleukocytes. In order to investigate whether the three antibodies preventbinding of P-selectin to PSGL-1, binding of purified human P-selectin toactivated T cells was measured in the presence of the three antibodies.KPL-1, known to block interaction of P-selectin and PSGL-1, was used asa positive control.

Human PBMC were activated with 1% PHA for 2 days and maintained inIL-2-containing medium for 3 days. The cells were incubated withtitrated 9F9, 15A7, 43B6, KPL-1 (an PSGL-1 antagonist), or a controlantibody (9E10) for 30 minutes, followed by the addition of recombinanthuman P-selectin (1.25 μg/ml). Binding of P-selectin to activated Tcells was measured by anti-P-selectin-FITC analyzed on FACS.

Consistent with previous reports, KPL-1 almost completely abolishedP-selectin's binding to activate T cells at a low concentration (0.31μg/ml). 43B6 blocked binding of P-selectin to activated T cells aseffectively as KPL-1 did, whereas a higher concentration of 9F9 wasrequired to achieve the same effect. Indeed, 0.08 mg/ml KPL or 43B6 wasneeded to abolish 50% of the binding. In contrast, 5 μg/ml 9F9 wasrequired. Moreover, 15A7 did not have any inhibitory effect onP-selectin binding even at 20 μg/ml. Surprisingly, it enhanced bindingof P-selectin to PSGL-1. These results indicate that 15A7 and P-selectinbind to different motifs of PSGL-1 on activated T cells.

The fact that 15A7 did not compete with P-selectin for PSGL-1 indicatesthat in vivo administration of 15A7 is not supposed to affect innateimmunity by interfering P-selectin-dependent recruitment of leukocytes.

It has been reported that PSGL-1 is expressed at low levels onplatelets. The effects of 15A7 antibodies on platelets were examined. Itwas found that the antibodies did not enhance or inhibit aggregation ofhuman platelets.

EXAMPLE 3 Hamster Monoclonal Antibody TAB4 Against Mouse PSGL-1

A monoclonal antibody against mouse PSGL-1, TAB4, was prepared in themanner similar to the method described in Example 1. It induced T cellapoptosis in vitro and depleted T cells in vivo. To determine if itinterfered with binding between mouse PSGL-1 and mouse P-selectin,competition analysis was performed in the manner similar to the methoddescribed in Example 2. It was found that TAB4 did not inhibit mouseP-selectin binding to mouse PSGL-1 even at a concentration as high as 20μg/ml.

EXAMPLE 4 Mouse Monoclonal Antibodies 4B7, 5C4, 12E7, 14B3, 17E5, and18D12

Additional monoclonal antibodies against human PSGL-1, 4B7, 5C4, 12E7,14B3, 17E5, and 18D12, were characterized. Upon binding to an activatedT cell, they all induced death of the activated T cells. Competitionanalysis was conducted in the manner described in Example 2 to determineif they blocked interaction between PSGL-1 and P-selectin. It was foundthat these antibodies have little, if any, inhibitory effect on humanP-selectin binding to human PSGL-1, even at the highest concentrationtested (5 μg/ml).

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of theinvention.

1. An isolated antibody that binds specifically to human P-SelectinGlycoprotein Ligand 1 (PSGL-1) without interfering with binding betweenPSGL-1 and P-Selectin, wherein the antibody, upon binding to humanPSGL-1 expressed on an activated T cell, induces death of the activatedT cell.
 2. The antibody of claim 1, wherein the antibody is a humanizedantibody.
 3. A composition comprising the antibody of claim
 1. 4. Thecomposition of claim 3, wherein the antibody is a humanized antibody.